1. Field of the Invention
This invention generally relates to processes for identifying, developing and using new biotypes of soilborne biocontrol agents. More specifically it relates to mutagenic processes for converting soilborne rhizosphere-incompetent biocontrol agents into rhizosphere-competent biocontrol agents. The term "rhizosphere competence" has been employed to describe an attribute of rhizobia characterized by their consistent association with legume root nodules. Here, we use the term to describe the ability of a microorganism to grow and function in the developing rhizosphere.
2. Description of the Art
The protection of plants from infection by soilborne fungal and bacterial pathogens by use of antagonistic microorganisms is well known to the art. For example, it is known that various Trichoderma spp. such as Trichoderma harzianum Rifai act as biological control agents against certain plant diseases. Nonetheless, use of Trichoderma spp., as biocontrol agents has been rather limited. This is mainly because seed treatment with Trichoderma spp. generally does not provide continued protection for the emerging root system of the maturing plant. Such seed treatment does serve to reduce preemergence damping-off but the root system is left unprotected. It is generally believed that this failure to protect the root system is because Trichoderma spp. are not rhizosphere-competent, see for example, Papavizas, G. C., Phytopathology 72: 121-125 (1982) and Chao, W. L., et al., Phytopathology 76: 60-65 (1986). Therefore, a need clearly exists for more effective methods for inducing biocontrol agents to colonize a plant's developing root system as well as its spermosphere.
This need has been partially met in the realm of certain bacterial biocontrol agents; see for example, Mendez-Castro, F. A. and Alexander, M., Method for establishing a bacterial inoculum on corn roots, Appl. Eviron., Microbiol. 45: 254-258 (1983). A similar plant protection strategy has been applied to fungal biocontrol agents (Ahmad, J. S., and Baker, R., Induction of rhizosphere competence in Trichoderma harzianum (Abstr.) Phytopathology 75: 1302 (1985)). This work indicated that when benomyl-tolerant mutants of Trichoderma harzianum Rifai were applied to seeds, the roots became colonized. However, the reason or reasons as to why such mutants are rhizosphere-competent, were not apparent. It seemed that many of the results obtained from following this research strategy were inconsistent and/or in conflict with what was then known about rhizosphere-competence. For example, it should be noted that the Papavizas article previously cited discloses the use of benomyl tolerant isolates of T. harzianum, obtained by ultraviolet light irradiation to test for rhizosphere-competence of bean and pea seedlings. However, Applicants found that ultraviolet mutants, tolerant to benomyl were not rhizosphere competent. Moreover, reports by other workers (see for example, Garrett, S. D., Pathogenic root-infected fungi, Cambridge University Press (1970)) postulated the theory that the share of a substrate obtained by any particular fungal species is determined partly by its intrinsic competitive saprophytic ability and partly by the balance between its inoculum potential and that of competing species. This report also theorized that production of, and tolerance to, antibiotics is another important attribute of successful rhizosphere fungi.
All of these theories were however, to some degree, inconsistent with the results of Applicants' rhizosphere competence tests. For example, Applicants found that both the mutants and the wild types had the same population density when applied to seeds. Moreover, none of Applicants' mutant strains have antibiotic activity in vitro except for a routing factor seen in the hyphal cytoplasm, at microscopic levels affecting Pythium spp. In trying to reconcile these theoretical and/or evidentiary conflicts, as well as those relating to the nature of the plant root surfaces themselves (see Foster, R. C., Rovira, A. D., and Cock, T. W., Ultrastructure of the Root-Soil Interface. Am. Phytopath. Soc., St. Paul, Minn. (1983)), Applicants postulated that rhizosphere-competence was somehow related to a possible possession by the mutant strains of enzymes for cellulase degradation. The subsequent finding by the Applicants that a wide variety of rhizosphere-competent Trichoderma mutant strains do in fact possess increased cellulase degradation capabilities, is a key aspect of the overall teachings of this disclosure.